Objectives of this research project are to define the basic structure of intervertebral disk proteoglycans, to examine the steps in the biosynthesis of proteoglycan monomer, assembly in the aggregate structures, and possible post-synthesis modification. Normal human and rabbit nucleus pulposus and annulus fibrosis will be studied. Proteoglycan extracted in the presence of protease inhibitors and purified under associative conditions will be fractionated into aggregating and nonaggregating proteoglycans by preparative Sepharose CL-2B chromatography and the fractions subsequently characterized by physical and biochemical techniques. The synthesis of the proteoglycan monomer and its subsequent modification will be studied initially in vivo using a rabbit or dog system with 35S-sulfate, 3H-serine, or glucosamine as proteoglycan precursors. These will be used to define structure of the initially synthesized molecule. The subsequent dissection of proteoglycan synthesis and assembly into aggregate structure could be performed with organ cultures of nucleus pulposus and annulus fibrosis, and in primary cultures of cells derived from nucleus and annulus. Proteoglycans labeled isotopically will be isolated by standard techniques and characterized in pulse-chase experiments to define the following parameters: the nature of the initially synthesized proteoglycan molecule and its substructure with respect to the hyaluronic acid binding site, keratan sulfate rich-region and chondroitin sulfate rich-region, as well as the possible presence of glycopeptides and its subsequent assembly into a stable aggregate. The nature of the assembly process will be pursued with pulse-chase experiments with isotopic precursors including: 35S-sulfate, 3H-serine, 3H-glucosamine, and specific competitive inhibitors. These inhibitors will include: cycohexamide or puromycin to inhibit protein synthesis, Beta-methylxyloside as a competitive inhibitor for chondroitin sulfate chain elongation, small hyaluronic acid (10-50 repeat disaccharides) to compete for assembly of the proteoglycan and link proteins into a stable aggregate. The molecules will be characterized by enzymatic, electrophoretic, and chromatographic techniques.